Dtsch. Entomol. Z. 68 (1) 2021, 179-188 | DOI 10.3897/dez.68.65540

eee oe
BERLIN

First instar nymphs of two peltoperlid stoneflies
(Insecta, Plecoptera, Peltoperlidae)

Shodo Mtow!?, Tadaaki Tsutsumi!

1 Faculty of Symbiotic Systems Science, Fukushima University, Kanayagawa 1, Fukushima, Fukushima 960-1296, Japan
http://zoobank.org/D39BD7BB-70F 9-4BA 6-83AB-15F4B81C57D9

Corresponding author: Shodo Mtow (impulse6 10@gmail.com)

Academic editor: Susanne Randolf # Received 11 March 2021 # Accepted 22 April 2021 Published 7 May 2021

Abstract

The first instar nymphs of two peltoperlid stoneflies, 1.e., Microperla brevicauda Kawai, 1958 of Microperlinae and Yoraperla uenoi
(Kohno, 1946) of Peltoperlinae, were examined and described. Additionally, the phylogeny and groundplan of the first instar nymphs
of Peltoperlidae and Plecoptera were considered. The first instar nymphs of 7. brevicauda have a slender body with a prognathous
head of typical shape; they represent a groundplan in Plecoptera. On the other hand, the first instar nymphs of Y. wenoi have a broad,
cockroach-like body with an orthognathous and shortened head, the latter being regarded as a potential autapomorphy of Peltoper-
linae. Such differences in body shape between the subfamilies are speculated to arise from heterochrony. The three-segmented cerci
of Y. uenoi are characteristic to Systellognatha, whereas the four-segmented cerci of M. brevicauda were independently acquired
within Microperlinae. The structure and distribution pattern of chloride cells in the first instar nymphs of Plecoptera were also dis-
cussed. The presence of coniform chloride cells 1s a potential groundplan of Arctoperlaria. One to two pairs of chloride cells are
distributed on the first nine abdominal segments of WM. brevicauda; this represents a groundplan character of Systellognatha. On the
other hand, one to four pairs of chloride cells are found on the second to ninth abdominal segments of Y. wenoi; this distribution

pattern may be an apomorphic groundplan of Peltoperlinae.

Key Words

Arctoperlaria, Systellognatha, Microperlinae, Peltoperlinae, phylogeny, chloride cell

Introduction

Plecoptera, commonly known as stoneflies, are a hemi-
metabolous, neopteran order containing approximately
3,700 described species with a worldwide distribution on
all continents except Antarctica (e.g., Zwick 1973; Fo-
chetti and Tierno de Figueroa 2008; DeWalt and Ower
2019). In terms of phylogenetic position, recent morpho-
logical, embryological, and molecular evidence supports
the placement of Plecoptera in the monophyletic group
Polyneoptera (e.g., Ishiwata et al. 2011; Yoshizawa 2011;
Mashimo et al. 2014; Misof et al. 2014; Wipfler et al.
2015, 2019; Song et al. 2016; Mtow and Machida 2018).
The relationships between family group taxa within Ple-
coptera, 1.e., 16 families, are widely accepted based on
the two-suborder concept (e.g., Zwick 2000; Beutel et al.

2014; McCulloch et al. 2016; Ding et al. 2019). The sub-
order Arctoperlaria mainly occurs in the Northern Hemi-
sphere, comprising 12 families within two subgroups,
Euholognatha and Systellognatha, which each contain
six families, 1.e., Scopuridae, Taeniopterygidae, Capni-
idae, Leuctridae, Nemouridae, and Notonemouridae of
Euholognatha, and Pteronarcyidae, Styloperlidae, Peltop-
erlidae, Perlidae, Chloroperlidae, and Perlodidae of Sys-
tellognatha. In contrast, the suborder Antarctoperlaria is
found only in the Southern Hemisphere and contains four
families, i.e., Eustheniidae, Diamphipnoidae, Austroperl-
idae, and Gripopterygidae.

Peltoperlidae is a systellognathan family present in
North America and East Asia; it contains almost 70 de-
scribed species (DeWalt and Ower 2019) and is comprised
of two subfamilies, Microperlinae and Peltoperlinae

Copyright Shodo Mtow, Tadaaki Tsutsumi. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

180 Shodo Mtow & Tadaaki Tsutsumi: First instar nymphs of two peltoperlid stoneflies

(Uchida and Isobe 1989; Zwick 2000). The monophy-
ly of Peltoperlidae and each subfamily is supported by
both morphological (e.g., Zwick 1973, 2000; Uchida and
Isobe 1989) and molecular phylogenetic evidence (Cao
et al. 2019). However, despite extensive research having
been conducted by multiple stonefly researchers, the sys-
tematic position of Peltoperlidae in Plecoptera remains
to be clarified. Groups that have been proposed as sister
group candidates are Pteronarcyidae (Mtow and Machida
2018), Styloperlidae (e.g., Uchida and Isobe 1989; Zwick
2000; Wang et al. 2017, 2019; Shen and Du 2020; Zhao
et al. 2020), Perlidae (e.g., Chen and Du 2017b; 2018;
Ding et al. 2019), Austroperlidae + Scopuridae (Illies
1965), Pteronarcyidae + Styloperlidae (Ding et al. 2019),
Styloperlidae + Perlidae (Chen and Du 2017a), Chlorop-
erlidae + Perlidae (Ding et al. 2019), Perloidea (= Perli-
dae + Chloroperlidae + Perlodidae) (Zwick 1973, 1980;
Nelson 1984; Thomas et al. 2000), Pteronarcyidae + Per-
loidea (e.g., Ricker 1952; Terry 2004; Kjer et al. 2006;
Shen and Du 2019; South et al. 2021), (Pteronarcyidae +
Chloroperlidae) + (Styloperlidae + Perlidae) (Chen and
Du 2017a), (Pteronarcyidae + Styloperlidae) + Perloidea
(Chen et al. 2018), (Taeniopterygidae + (Nemouridae +
Notonemouridae)) + (Leuctridae + Capniidae) (Ricker
1950), ((Eusthentidae + Diamphipnoidae) + Austroperl-
idae) + (Gripopterygidae + (Capniidae + (Scopuridae +
(Nemouridae + Notonemouridae)))) (Terry and Whiting
2005), and (Pteronarcyidae + Perloidea) + ((Taenioptery-
gidae + Leuctridae) + ((Scopuridae + Notonemouridae) +
(Capniidae + Antarctoperlaria))) (Kjer et al. 2006).

It has previously been suggested that studies of Plecop-
tera first instar nymphs could be a potential source of phylo-
genetic information that could contribute to clarifying phy-
logenetic relationships (Harper 1979; Sephton and Hynes
1982). To date, the data collected in this area has been frag-
mentary and a detailed study has yet to be conducted. In ad-
dition, while the taxonomy and morphology of adults, older
nymphs, and egg structures from Peltoperlidae have been
studied extensively (e.g., Stark and Stewart 1981; Uchida
and Isobe 1988, 1989; Stark and Nelson 1994; Stark and
Sivec 2000, 2007; Stark et al. 2015; Chen 2020), informa-
tion on peltoperlid hatchlings is entirely lacking.

Given this background, in the present study we ex-
amined and described, for the first time, the first instar
nymphs of two Japanese peltoperlids, 1.e., Microperla
brevicauda Kawai, 1958 (Kawai 1958) (Fig. 1A) of Mi-
croperlinae and Yoraperla uenoi (Kohno, 1946) (Kohno
1946) (Fig. 1B, C) of Peltoperlinae, as two representative
species. We compared the data obtained to that from pre-
vious studies on other plecopterans with the aim of dis-
cussing the groundplan and phylogeny of Peltoperlidae
within Plecoptera as well as reconstructing the ground-
plan of their first instar nymphs.

Methods

Females of Microperla brevicauda and Yoraperla ue-
noi were collected from Japane Nara, Higashi yoshi-

dez.pensoft.net

no, a tributary of the Shigo river; alt. 420 m; around
34°22.67'N, 136°01.80'E; 13 Apr. 2016, and Japans
Nagano, Ueda, Kara-sawa stream; alt. 1220 m; around
36°31. TON: 138°20:26'E;- 12) Jul. 2019, grespectively.
They were kept separately at 12°C in plastic cases (68
mm x 39 mm x 15 mm) containing tissue paper and fed
on Mitani Mushi-jelly, i.e., commercial food for insects
(Fig. 1C). The first instar nymphs were obtained from
eggs deposited and incubated in plastic cases (36 mm
x 36 mm x 14 mm) filled with water at 12°C. These
were then fixed with either Bouin’s fixative (saturated
picric acid aqueous solution : formalin : acetic acid =
15:5: 1) or Kahle’s fixative (ethyl alcohol : formalin
: acetic acid : distilled water = 15 : 6: 2: 30) for 24h
and stored in 80% ethyl alcohol at room temperature.
The following measurements were taken from the fixed
nymphs: (1) body length (from the top of head to the
tip of abdomen), (2) antennal length, (3) length and (4)
width of head, (5) length and (6) width of pronotum, (7)
pronotum width/body length ratio, (8) abdominal width,
and (9) cercus length.

To observe chloride cells, some fixed specimens were
stained with Mayer’s acid haemalum for 1 h, mounted in
distilled water, examined using an Olympus BX43 bio-
logical microscope, and finally photographed with a Pen-
tax K-70 camera. Other fixed specimens were dehydrated
in a graded ethanol series, immersed in acetone, and em-
bedded in a Kulzer Technovit 7100 methacrylate resin in
accordance with the protocol described by Machida et al.
(1994). Serial, semi-thin sections at a thickness of 2 um
were cut using a Leica RM2235 semi-thin microtome
equipped with a Leica TC-65 tungsten carbide knife. Sec-
tions were then stained with Mayer’s acid haemalum for
1 h, 1% eosin Y for 1 h, and 1% fast green FCF 100% eth-
anol solution for 1 min, before being observed under the
Olympus BX43 biological microscope and photographed
with the Pentax K-70 camera.

For scanning electron microscopy, the fixed specimens
were dehydrated in a graded ethanol series, naturally
dried with HMDS (1,1,1,3,3,3-Hexamethyldisilazane) as
described by Faull and Williams (2016), mounted on a
stab, and then observed under a Hitachi TM-1000 scan-
ning electron microscope at 15 kV without coating. Some
mounted specimens were examined under the Olympus
BX43 biological microscope and photographed with the
Pentax K-70 camera.

The specimens examined in the present study have
been deposited in the collection of the Faculty of Symbi-
otic Systems Science, Fukushima University.

Results

The present study follows the view of Matsuda (1976) on the
abdominal segmentation on Plecoptera, 1.e., that the sternum
of the first abdominal segment is greatly reduced or absent.
The definition of chloride cells, which are divided morpho-
logically into four types, 1.e., caviform, coniform, bulbiform,
and floriform, follows that of Wichard et al. (1999).

Dtsch. Entomol. Z. 68 (1) 2021, 179-188

Figure 1. Adults of Microperla brevicauda and Yoraperla uenoi. A. M. brevicauda; B. Y. uenoi; C. Y. uenoi eating a commercial

food (star) in a plastic case.

Table 1. Measurements of the fixed specimens of first instar
nymphs of Microperla brevicauda and Yoraperla uenoi.

Microperla brevicauda Yoraperla uenoi

Specimens examined 5 5
Body length (um) 623.54 12.3 574.1 + 6.0
Antennal length (um) 402.3 + 53.5 282.3 432.2
Head length (um) 115.34 10.9 96.5 + 6.0
Head width (um) 180.0 42.9 217.6483
Pronotum length (um) 72.9 £ 6.0 82.4+ 6.4
Pronotum width (um) 171.8444 236.5 + 12.0
Pronotum width / Body length 0.27+0.01 0.41 + 0.02
Abdominal width (um) 114.14+2.9 156.47 42.9
Cercus length (um) 195.3 + 14.6 160.0 + 6.9

Measurements of the first instar nymphs of Microperla
brevicauda and Yoraperla uenoi are shown in Table 1.

Microperla brevicauda Kawai, 1958
Figs 2A-D, 4A, B, D, E

Description. Body slender, uniformly white, sparsely
covered by long and short fine setae, without gill and
ocelli (Fig. 2A, B). Head prognathous, subtriangular
(Fig. 2A, B). Antenna nine-segmented, longer than two-
thirds of body length (Fig. 2A, B; Table 1). Compound
eye reddish-black with four ommatidia. Labrum near-

ly semicircular, covering part of mandible (Fig. 2C).
Maxillary coxopodites divided into distal cardo and
proximal stipes (Fig. 2C); maxillary palp and endites
of maxilla and lateral galea well developed, but mesal
lacinia hardly visible externally (Fig. 2C). Labial coxo-
podites divided into proximal postmentum and distal
prementum, but hardly recognizable in fixed specimen
(Fig. 2C); labial palp and endites of labium, lateral para-
glossa, and mesal glossa well developed (Fig. 2C). Pro-
notum rectangular, almost same width as the head, and
wider than the abdomen (Fig. 2A; Table 1). Lateral mar-
gin of mesonotum and metanotum less developed (Fig.
2A). Thoracic appendage consisting of coxa, trochanter,
femur, tibia, tarsus with three tarsomeres, and pretarsus
with ungues (Fig. 2D); tibia longer than femur (Fig.
2D). Abdominal segments with a row of long fine setae
along posterior margin of each tergum (Figs 2A, 4A).
Coniform chloride cells approximately 10 um in diame-
ter and 1 um in height distributed on lateral side of first
nine abdominal segments; one pair on first, eighth, and
ninth targa; two pairs on second to seventh segments,
i.e., one pair on each of second to seventh terga and ster-
na (Fig. 4A, B, D, E). Less sclerotized supraanal lobe on
hind margin of tenth tergum (Fig. 2A). Cerci four-seg-
mented, with a crown of long and short fine setae on

dez.pensoft.net

Shodo Mtow & Tadaaki Tsutsumi: First instar nymphs of two peltoperlid stoneflies

Figure 2. First instar nymphs of Microperla brevicauda. A. Habitus, dorsal view, scanning electron microscopy (SEM); B. Habitus,
dorsal view, same specimen as in (A), light microscopy; C. Mouth parts, ventral view, SEM; D. Middle leg, SEM. Abbreviations:
An, antenna; Ca, cardo; Ce, cercus; Cx, coxa; Fe, femur; Ga, galea; Gl, glossa; H, head; LbP, labial palp; Lr, labrum; Md, mandible;
Msn, mesonotum; Mtn, metanotum; Pel, paraglossa; Pta, pretarsus; Spa, supraanal lobe; St, stipes; Ta, tarsus; T1, tibia; Tr, trochan-
ter. Scale bars: 100 um (A, B); 20 um (C, D).

dez.pensoft.net

Dtsch. Entomol. Z. 68 (1) 2021, 179-188

posterior margin of first three segments; short fine setae
at apex of fourth segment (Fig. 2A).

Yoraperla uenoi (Kohno, 1946)
Figs 3A-D, 4C, F, G

Description. Body broad and slightly cockroach-like,
uniformly white, covered by brownish, long and short,
stout setae, without gill and ocelli (Fig. 3A, B). Head
orthognathous, trapezoidal, and highly shortened
(Fig. 3A). Antenna nine-segmented, same as half of
body length (Fig. 3A, B; Table 1). Compound eye red-
dish-black but ommatidia inconspicuous in fixed spec-
imens; according to Mtow and Machida (2018), four
ommatidia formed in full-grown embryos of Y. uenoi.
Labrum slightly trapezoidal, covering part of mandible
(Fig. 3C). Mandible well-developed with teeth at its
apex (Fig. 3C). Maxillary coxopodites divided into dis-
tal cardo and proximal stipes, but latter hardly visible
from ventral view (Fig. 3C); maxillary palp and endites
of maxilla, lateral galea, and mesal lacinia well devel-
oped (Fig. 3C); teeth formed at tip of lacinia (Fig. 3C).
Labial coxopodites divided into proximal postmentum
and distal prementum, but hardly recognizable in fixed

183

specimen (Fig. 3C); labial palp and endites of labium,
lateral paraglossa, and mesal glossa well developed
(Fig. 3C). Pronotum rectangular with corners slightly
rounded, slightly wider than head and wider than ab-
domen (Fig. 3A; Table 1). Mesonotum and metanotum
trapezoidal, slightly widening posteriorly (Fig. 3A).
Thoracic appendage consisting of coxa, trochanter, fe-
mur, tibia, tarsus with three tarsomeres, and pretarsus
with ungues (Fig. 3D); tibia almost identical in length
to femur (Fig. 3D); two claws can be recognized from
ventral view (data not shown). Abdominal segments
with a row of long and short stout setae along posterior
margin of each tergum, except first to third terga, cov-
ered by metanotum (Figs 3A, 4F):; first terga without
setae, second and third tergum with setae barely visible
in section (data not shown). Coniform chloride cells
approximately 10 um in diameter and 1.5 um in height
distributed on posterior margin of second to ninth ab-
dominal segments; one pair on second sternum; three
pairs on third sternum; four pairs on fourth to seventh
sterna; three or four pairs on eighth sternum; two pairs
on ninth sternum (Fig. 4C, F, G). Cerci three-segment-
ed, with a crown of long and short stout setae on pos-
terior margin of first two segments; short stout setae on
apex of third segment (Fig. 3A).

Figure 3. First instar nymphs of Yoraperla uenoi. A. Habitus, dorsal view, scanning electron microscopy (SEM); B. Habitus, dorsal

view, Same specimen as in (A), light microscopy; C. Mouth parts, ventral view, SEM, right side of maxillary pulp artificially lack-

ing; D. Middle leg, SEM. Abbreviations: An, antenna; Ce, cercus; Cx, coxa; Fe, femur; Ga, galea; Gl, glossa; H, head; La, lacinia;

LbP, labial palp; Lr, labrum; Md, mandible; Msn, mesonotum; Mtn, metanotum; Pel, paraglossa; Pta, pretarsus; St, stipes; Ta, tarsus;

Ti, tibia; Tr, trochanter. Scale bars: 100 um (A, B); 20 um (C, D).

dez.pensoft.net

184 Shodo Mtow & Tadaaki Tsutsumi: First instar nymphs of two peltoperlid stoneflies

Figure 4. Chloride cells of first instar nymphs of Microperla brevicauda and Yoraperla uenoi, anterior to the left. A. Abdomen of /.
brevicauda, lateral view, stained with Mayer’s acid haemalum; B, C. Horizontal sections of fifth abdominal segment of M. brevicau-
da (B) and Y. uenoi (C); D, E. Abdomen of M. brevicauda, lateral view, scanning electron microscopy (SEM), all abdominal seg-
ments (D) and enlargement of chloride cells (E); F, G. Abdomen of Y. wenoi, ventrolateral view, SEM, all abdominal segments (F)
and enlargement of chloride cells (G). Arrowheads show the chloride cells. Abbreviations: Al, 2, 3, 5, and 10: first, second, third,
fifth and tenth abdominal segments, respectively; Ce, cercus; Mtn, metanotum. Scale bars: 50 um (A, D, F); 10 um (B, C, E, G).

dez.pensoft.net

Dtsch. Entomol. Z. 68 (1) 2021, 179-188

Discussion

The first instar nymphs of Microperla brevicauda of Mi-
croperlinae can be characterized by a slender body and
typical head, 1.e., being prognathous and subtriangular
in shape. These features are predominant in Plecop-
tera, 1.e., Antarctoperlaria: Eustheniidae (Helson 1935;
Sephton and Hynes 1982), Austroperlidae (Sephton and
Hynes 1982), and Gripopterygidae (Sephton and Hynes
1982); Arctoperlaria: Euholognatha, Scopuridae (Komat-
su 1956; Mtow 2019), Taeniopterygidae (e.g., Berthéle-
my 1979; Harper 1979), Leuctridae (e.g., Harper 1979;
Snellen and Stewart 1979a), Capniidae (Harper 1979),
Nemouridae (e.g., Harper 1979), and Notonemouridae
(Sephton and Hynes 1982); Systellognatha: Pteronarcy-
idae (Miller 1939), Perlidae (e.g., Harper 1979; Kishimo-
to and Ando 1985), Chloroperlidae (Harper 1979), and
Perlodidae (Berthélemy 1979; Harper 1979). Therefore,
we conclude that these characters represent a groundplan
of Plecoptera. In contrast, the first instar nymph of Yor-
aperla uenoi of Peltoperlinae can be characterized by a
broad, slightly cockroach-like body and modified head,
i.e., being orthognathous, shortened, and trapezoidal in
shape. The head shape is unique to Peltoperlinae with-
in Plecoptera and could be a potential autapomorphy of
this group, which is consistent with the understanding of
Zwick (2000), 1.e., that a modified head shape such as this
may be an apomorphic groundplan of Peltoperlinae.

Notably, the cockroach-like body shape, which is
regarded as an autapomorphy of Peltoperlidae (Zwick
1973, 2000; Uchida and Isobe 1989), was found in the
first instar nymphs of Peltoperlinae but did not appear in
those of Microperlinae. Given that the older nymphs of
M. brevicauda have much broader, cockroach-like bodies
(e.g., Shimizu et al. 2005) and that the full-grown embry-
os of Y. uenoi acquire the configuration of the first instar
nymphs (Mtow and Machida 2018), such differences in
the body shape of peltoperlid first instar nymphs could
be interpreted as a result of heterochrony. In other words,
morphogenesis of the cockroach-like body shape may
commence by the later embryonic period in Peltoperlinae
but occur only during the postembryonic stages in Micro-
perlinae. Further detailed examination of the embryonic
and postembryonic development of Peltoperlidae will be
required to broaden our knowledge of nymphal shape
morphogenesis in this family.

The present study revealed that the first instar nymphs
of M. brevicauda and Y. uenoi have four-segmented and
three-segmented cerci, respectively. In Systellognatha,
three-segmented cerci are found predominantly in Pter-
onarcyidae (Miller 1939), Perlidae (e.g., Khoo 1964;
Harper 1979; Kishimoto and Ando 1985), Chloroperlidae
(Khoo 1964; Harper 1979), and Perlodidae (e.g., Khoo
1964; Harper 1979) with two exceptional cases, which
have four cercal segments: Perlesta placida (Hagen) of
Perlidae (Snellen and Stewart 1979b) and Hydroperla
crosbyi (Needham & Claassen) of Perlodidae (Ober-
ndorfer and Stewart 1977). Therefore, the sharing of
three-segmented cerci, which are found in Y. uwenoi of

185

Peltoperlinae, might be characteristic to Systellognatha,
whereas four cercal segments, such as those found in
M. brevicauda, were most likely acquired independently
within Microperlinae.

The chloride cells, which are known to have os-
moregulatory functions (e.g., Wichard et al. 1999), of
Antarctoperlaria nymphs are floriform type only (e.g.,
Zwick 1973), even in the first nymphal stage (Sephton
and Hynes 1982), which is regarded as an apomorphic
groundplan of Antarctoperlaria (e.g., Zwick 1973, 2000).
On the other hand, the presence of three other types of
chloride cells, 1.e., caviform, coniform, and bulbiform,
has been observed in arctoperlarian nymphs (e.g., Wich-
ard et al. 1999; Tamura and Kishimoto 2010), but only the
coniform type has been observed in hatchlings of Arcto-
perlaria (Berthélemy 1979; Kishimoto and Ando 1985)
with the exception of euholognathan Notonemouridae,
which has bulbiform type (Sephton and Hynes 1982). In
the present study, the first instar nymphs of M. brevicauda
and Y. uenoi had chloride cells of coniform type. Thus,
the type of chloride cells found in M. brevicauda and
Y. uenoi is apparently comparable to those found in euho-
lognathan Brachyptera braueri Klapalek of Taenioptery-
gidae (Berthélemy 1979), as well as systellognathan Ka-
mimuria tibialis (Pictet) of Perlidae (Kishimoto and Ando
1985) and Perlodes microcephalus (Pictet) of Perlodidae
(Berthélemy 1979). Therefore, the coniform type of chlo-
ride cells may be regarded as a potential groundplan of
Arctoperlaria. The bulbiform type of chloride cells found
in Notonemouridae might be due to a secondary modifi-
cation from the coniform type, because as Wichard et al.
(1999) pointed out, the coniform type is regarded as the
basic type of chloride cells.

In the present study, we also distinguished two dis-
tribution types of chloride cells in Peltoperlidae: (1) the
first type, in which one to two pairs of chloride cells are
distributed on the first nine abdominal segments, is found
in M. brevicauda of Microperlinae; (2) the second type,
in which one to four pairs of chloride cells are distribut-
ed on the second to eighth abdominal segments, is found
in Y. uenoi of Peltoperlinae. Additional examination of
chloride cells will be required to cover more lineages of
Plecoptera in detail. However, given the distribution of
the chloride cells on the abdomen of first instar nymphs,
it may be meaningful that the first type has also been ob-
served in Perlidae (Kishimoto 1983; Kishimoto and Ando
1985) and Perlodidae (Berthélemy 1979). Therefore, we
could postulate the following scenario: (1) the first type
is a groundplan character in Systellognatha, and (2) this
groundplan feature was inherited by Microperlinae in
Peltoperlidae; whereas, (3) the second type was acquired
by Peltoperlinae as an apomorphic groundplan.

Conclusion
In the present study, we (1) examined and described the
first instar nymphs of Peltoperlidae for the first time,

(2) reconstructed the groundplan of first instar nymphs

dez.pensoft.net

186 Shodo Mtow & Tadaaki Tsutsumi: First instar nymphs of two peltoperlid stoneflies

from Peltoperlidae and Plecoptera, and (3) demonstrated
that data collected from first instar nymphs could pro-
vide a new basis for discussion and reconstruction of the
groundplan and phylogeny of Plecoptera. To improve un-
derstanding of the plecopteran groundplan and phylogeny
further, more detailed studies of first instar nymphs must
be conducted; these should consider all major lineages
of Plecoptera, especially the antarctoperlarian Diamphip-
noidae and arctoperlarian Styloperlidae, on which infor-
mation is entirely lacking.

Acknowledgements

We are grateful to Ms. Mitsuki Mtow for her kind assis-
tance with the collection of materials; Drs. Susanne Ran-
dolf and Peter Zwick for their helpful comments; ENA-
GO (www.enago.jp) for the English-language review. The
present study was supported by a Sasakawa Scientific Re-
search Grant from The Japan Science Society (28-515)
and by a JSPS (Japan Society for the Promotion of Sci-
ence) KAKENHI: Grant-in-Aid for JSPS Research Fel-
low, Grant number JP18J10360 and JP20J00039 to SM.

References

Berthélemy C (1979) Mating, incubation period of the eggs, and first
larval stages of Brachyptera braueri and Perlodes microcephalus
(Plecoptera). Annales de Limnologie 15(3): 317-335. https://dot.
org/10.1051/limn/1979015

Beutel RG, Friedrich F, Ge SQ, Yang XK (2014) Insect Morpholo-
gy and Phylogeny. Walter de Gruyter, Berlin, 516 pp. https://dot.
org/10.1515/9783 110264043

Cao JJ, Wang Y, Wei X, Chen S, Li WH (2019) First mitochondrial
genome of a stonefly from the subfamily Microperlinae: Microperla
geei (Plecoptera: Peltoperlidae). Mitochondrial DNA Part B 4(2):
2679-2680. https://doi.org/10.1080/23802359.2019. 1644234

Chen ZT (2020) Holomorphology and neotype designation of Microp-
erla geei Chu, with egg morphology of Microperla ginlinga Chen
(Plecoptera: Peltoperlidae). Zootaxa 4780(3): 563-578. https://doi.
org/10.11646/zootaxa.4780.3.8

Chen ZT, Du YZ (2017a) Complete mitochondrial genome of Capnia
zijinshana (Plecoptera: Capniidae) and phylogenetic analysis among
stoneflies. Journal of Asia-Pacific Entomology 20(2): 305-312.
https://doi.org/10.1016/j.aspen.2017.01.013

Chen ZT, Du YZ (2017b) First mitochondrial genome from Nemouridae
(Plecoptera) reveals novel features of the elongated control region
and phylogenetic implications. International Journal of Molecular
Sciences 18(5): 996. https://doi.org/10.3390/1jms18050996

Chen ZT, Du YZ (2018) The first two mitochondrial genomes from Tae-
niopterygidae (Insecta: Plecoptera): structural features and phylo-
genetic implications. International Journal of Biological Macromol-
ecules 111: 70-76. https://doi.org/10.1016/j.1jbiomac.2017.12.150

Chen ZT, Zhao MY, Xu C, Du YZ (2018) Molecular phylogeny of Sys-
tellognatha (Plecoptera: Arctoperlaria) inferred from mitochondrial
genome sequences. International Journal of Biological Macromole-
cules 111: 542-547. https://doi.org/10.1016/j.ijbiomac.2018.01.065

dez.pensoft.net

DeWalt RE, Ower GD (2019) Ecosystem services, global diversity, and
rate of stonefly species descriptions (Insecta: Plecoptera). Insects
10(4): 99. https://doi.org/10.3390/insects 10040099

Ding S, Li W, Wang Y, Cameron SL, Muranyi D, Yang D (2019) The
phylogeny and evolutionary timescale of stoneflies (Insecta: Ple-
coptera) inferred from mitochondrial genomes. Molecular Phylo-
genetics and Evolution 135: 123-135. https://doi.org/10.1016/,.
ympev.2019.03.005

Faull KJ, Williams CR (2016) Differentiation of Aedes aegypti and Ae-
des notoscriptus (Diptera: Culicidae) eggs using scanning electron
microscopy. Arthropod Structure & Development 45(3): 273-280.
https://doi.org/10.1016/j.asd.2016.01.009

Fochetti R, Tierno de Figueroa JM (2008) Global diversity of stoneflies
(Plecoptera; Insecta) in freshwater. Hydrobiologia 595: 365-377.
https://doi.org/10.1007/s10750-007-903 1-3

Harper PP (1979) Observations on the early instars of stoneflies (Ple-
coptera). Gewasser und Abwasser 64: 18-28.

Helson GAH (1935) The hatching and early instars of Stenoperla pra-
sina Newman. Transactions and Proceedings of the Royal Society
of New Zealand 65: 11—16. https://paperspast.natlib.govt.nz/period-
icals/TPRSNZ1936-65.2.6.3

Illies J (1965) Phylogeny and zoogeography of the Plecoptera. Annual
Review of Entomology 10: 117-140. https://doi.org/10.1146/an-
nurev.en.10.010165.001001

Ishiwata K, Sasaki G, Ogawa J, Miyata T, Su ZH (2011) Phylogenet-
ic relationships among insect orders based on three nuclear pro-
tein-coding gene sequences. Molecular Phylogenetics and Evolution
58(2): 169-180. https://doi.org/10.1016/j.ympev.2010.11.001

Kawai T (1958) Studies on the holognathous stoneflies of Japan IV
Description of a new species of the genus Microperla (Peltop-
erlidae). Kontya 26(3): 138-141. https://dl.ndl.go.jp/info:ndljp/
pid/10649704

Khoo SG (1964) Studies on the biology of stoneflies. PhD Thesis, Uni-
versity of Liverpool, Liverpool, 161 pp. https://ethos.bl.uk/Order-
Details.do?uin=uk.bl.ethos.503474

Kishimoto T (1983) Structure and distribution of the chloride cells in
the larvae of Kamimuria tibialis Pictet (Insecta: Plecoptera). Pro-
ceedings of Arthropodan Embryological Society of Japan 19: 16.
http://aesj.co-site.jp/num19/1983_Vol.19_16.pdf

Kishimoto T, Ando H (1985) External features of the developing embryo
of the stonefly, Kamimuria tibialis (Pictet) (Plecoptera, Perlidae).
Journal of Morphology 183(3): 311-326. https://doi.org/10.1002/
jmor. 1051830308

Kjer KM, Carle FL, Litman J, Ware J (2006) A molecular phylog-
eny of Hexapoda. Arthropod Systematics & Phylogeny 64(1):
35-44. https://www.senckenberg.de/wp-content/uploads/2019/08/
asp 64 1 kjer et_al 35-44. pdf

Kohno M (1946) Corrigenda to of my paper “My observation of genus

Nogiperla and genus Peltoperla with description of new genus Neo-
pltoperla (Order Plecoptera). Kontyt Sekai (Insect World) 50(574):
11-14. [In Japanese]

Komatsu T (1956) On the imago, egg and first instar of Scopura longa
Ueno. New Entomologist 5: 13—21. [In Japanese with English abstract]

Machida R, Nagashima T, Ando H (1994) Embryonic development of
the jumping bristletail Pedetontus unimaculatus Machida, with spe-
cial reference to embryonic membranes (Hexapoda: Microcoryphia,
Machilidae). Journal of Morphology 220(2): 147-165. https://doi.
org/10.1002/;mor. 1052200205

Dtsch. Entomol. Z. 68 (1) 2021, 179-188

Mashimo Y, Beutel RG, Dallai R, Lee CY, Machida R (2014) Embry-
onic development of Zoraptera with special reference to external
morphology, and its phylogenetic implications (Insecta). Journal of
Morphology 275(3): 295-312. https://doi.org/10.1002/jmor.20215

Matsuda R (1976) Morphology and Evolution of the Insect Abdomen.
Pergamon Press, Oxford, 544 pp. https://doi.org/10.1016/C2013-0-
05688-5

McCulloch GA, Wallis GP, Waters JM (2016) A time-calibrated phylog-
eny of southern hemisphere stoneflies: testing for Gondwanan ori-
gins. Molecular Phylogenetics and Evolution 96: 150-160. https://
doi.org/10.1016/j.ympev.2015.10.028

Miller A (1939) The egg and early development of the stonefly, Pzero-
narcys proteus Newman (Plecoptera). Journal of Morphology 64(3):
555-609. https://doi.org/10.1002/jmor. 1050640308

Misof B, Liu S, Meusemann K, Peters RS, Donath A, Mayer C, Frand-
sen PB, Ware J, Flouri T, Beutel RG, Niehuis O, Petersen M, Izqui-
erdo-Carrasco F, Wappler T, Rust J, Aberer AJ, Aspéck U, Aspock H,
Bartel D, Blanke A, Berger S, Bohm A, Buckley TR, Calcott B, Chen
J, Friedrich F, Fukui M, Fujita M, Greve C, Grobe P, Gu S, Huang
Y, Jermiin LS, Kawahara AY, Krogmann L, Kubiak M, Lanfear R,
Letsch H, Li Y, Li Z, Li J, Lu H, Machida R, Mashimo Y, Kapli P,
McKenna DD, Meng G, Nakagaki Y, Navarrete-Heredia JL, Ott M,
Ou Y, Pass G, Podsiadlowski L, Pohl H, von Reumont BM, Schiitte
K, Sekiya K, Shimizu S, Slipinski A, Stamatakis A, Song W, Su X,
Szucsich NU, Tan M, Tan X, Tang M, Tang J, Timelthaler G, To-
mizuka S, Trautwein M, Tong X, Uchifune T, Walz] MG, Wiegmann
BM, Wilbrandt J, Wipfler B, Wong TKF, Wu Q, Wu G, Xie Y, Yang
S, Yang Q, Yeates DK, Yoshizawa K, Zhang Q, Zhang R, Zhang W,
Zhang Y, Zhao J, Zhou C, Zhou L, Ziesmann T, Zou S, Li Y, Xu X,
Zhang Y, Yang H, Wang J, Wang J, Kjer KM, Zhou X (2014) Phylog-
enomics resolves the timing and pattern of insect evolution. Science
346(6210): 763-767. https://dot.org/10.1126/science. 1257570

Mtow S (2019) Comparative embryology of Arctoperlaria (Insecta:
Plecoptera). PhD Thesis, University of Tsukuba, Tsukuba, 186 pp.
https://doi.org/10.15068/00156438

Mtow S, Machida R (2018) Egg structure and embryonic development
of arctoperlarian stoneflies: a comparative embryological study (Ple-
coptera). Arthropod Systematics & Phylogeny 76(1): 65—86. https://
www.senckenberg.de/wp-content/uploads/2019/07/05_asp_76-1_
mtow_65-86.pdf

Nelson CH (1984) Numerical cladistic analysis of phylogenetic rela-
tionships in Plecoptera. Annals of the Entomological Society of
America 77(4): 466-473. https://doi.org/10.1093/aesa/77.4.466

Oberndorfer RY, Stewart KW (1977) The life cycle of Hydroperla cros-
byi (Plecoptera: Perlodidae). Great Basin Naturalist 37(2): 260-273.
https://scholarsarchive.byu.edu/gbn/vol37/iss2/12

Ricker WE (1950) Some evolutionary trends in Plecoptera. Proceedings
of the Indiana Academy of Science 59: 197-209. http://journals.iu-
pui.edu/index.php/1as/article/view/567 1

Ricker WE (1952) Systematic Studies in Plecoptera. Indiana University
Publications, Science Series No. 18, 200 pp.

Sephton DH, Hynes HBN (1982) Observations on the first instar
nymphs of several Australian stoneflies (Plecoptera). Aquatic In-
sects 4(4): 237-252. https://doi.org/10.1080/01650428209361110

Shen Y, Du YZ (2019) The mitochondrial genome of Leucira sp. (Ple-
coptera: Leuctridae) and its performance in phylogenetic anal-
yses. Zootaxa 4671(4): 571-580. https://doi.org/10.11646/zoot-
axa.4671.4.8

187

Shen Y, Du YZ (2020) The complete mitochondrial genome of Flavoperla
biocellata Chu, 1929 (Plecoptera: Perlidae) and the phylogenetic anal-
yses of Plecoptera. PeerJ 8: e8762. https://doi.org/10.7717/peerj.8762

Shimizu T, Inada K, Uchida S (2005) Plecoptera. In: Kawai T, Tanida K
(Eds) Aquatic Insects of Japan: Manual with Keys and Illustration.
Tokai University Press, Hadanoshi, 237—290. [in Japanese]

Snellen RK, Stewart KW (1979a) The life cycle and drumming behav-
ior of Zealeuctra claasseni (Frison) and Zealeuctra hitei Ricker and
Ross (Plecoptera: Leuctridae) in Texas, USA. Aquatic Insects 1(2):
65-89. https://doi.org/10.1080/01650427909360980

Snellen RK, Stewart KW (1979b) The life cycle of Perlesta placida
(Plecoptera: Perlidae) in an intermittent stream in Northern Texas.
Annals of the Entomological Society of America 72(5): 659-666.
https://doi.org/10.1093/aesa/72.5.659

Song N, Li H, Song F, Cai W (2016) Molecular phylogeny of Polyneop-
tera (Insecta) inferred from expanded mitogenomic data. Scientific
Reports 6: 36175. https://doi.org/10.1038/srep36175

South EJ, Skinner RK, DeWalt RE, Kondratieff BC, Johnson KP, Davis
MA, Lee JJ, Durfee RS (2021) Phylogenomics of the North Ameri-
can Plecoptera. Systematic Entomology 46(1): 287-305. https://doi.
org/10.1111/syen.12462

Stark BP, Stewart KW (1981) The Nearctic genera of Peltoperlidae
(Plecoptera). Journal of the Kansas Entomological Society 54(2):
285-311. https://www,jstor.org/stable/25084161

Stark BP, Nelson CR (1994) Systematics, phylogeny and zoo-
geography of genus Yoraperla (Plecoptera: Peltoperlidae).
Insect Systematics & Evolution 25(3): 241-273. https://doi.
org/10.1163/18763 129400072

Stark BP, Sivec I (2000) Redescription of Microperla geei Chu (Plecop-
tera: Peltoperlidae). Acta Entomologica Slovenica 8(2): 101-106.
http://www.dlib.si/7 URN=URN:NBN:SI:DOC-GP5AP1D6

Stark BP, Sivec I (2007) New species and records of Asian Peltoperlidae
(Insecta: Plecoptera). Iliesia 3(12): 104-126. http://lliesia.species-
file.org/papers/Illiesia03-12.pdf

Stark BP, Kondratieff BC, Sandberg JB, Gill BA, Verdone CJ, Harrison
AB (2015) Sierraperla Jewett 1954 (Plecoptera: Peltoperlidae), dis-
tribution, egg morphology and description of a new species. Illiesia
11(02): 8-22. http://lliesia.speciesfile.org/papers/Illiesia 11-02. pdf

Tamura F, Kishimoto T (2010) Morphology and distribution of the
chloride cells in the nymphal legs of Plecoptera. Bulletin of Center
for Natural Environmental Education, Nara University of Educa-
tion 11: 9-24. [In Japanese with English abstract] http://hdl-handle.
net/10105/3235

Terry MD (2004) Phylogeny of the polyneopterous insects with em-
phasis on Plecoptera: molecular and morphological evidence. PhD
Thesis, Brigham Young University, Provo, 118 pp. https://scholar-
sarchive.byu.edu/etd/1134/

Terry MD, Whiting MF (2005) Mantophasmatodea and phylogeny of
the lower neopterous insects. Cladistics 21(3): 240-257. https://doi.
org/10.1111/7.1096-003 1.2005.00062.x

Thomas MA, Walsh KA, Wolf MR, McPheron BA, Marden JH (2000)
Molecular phylogenetic analysis of evolutionary trends in stone-
fly wing structure and locomotor behavior. Proceedings of the
National Academy of Sciences 97(24): 13178-13183. https://doi.
org/10.1073/pnas.230296997

Uchida S, Isobe Y (1988) Cryptoperla and Yoraperla from Japan and
Taiwan (Plecoptera: Peltoperlidae). Aquatic Insects 10(1): 17-31.
https://doi.org/10.1080/01650428809361306

dez.pensoft.net

188 Shodo Mtow & Tadaaki Tsutsumi: First instar nymphs of two peltoperlid stoneflies

Uchida S, Isobe Y (1989) Styloperlidae, stat. nov. and Microperlinae,
subfam. nov. with a revised system of the family group Systellogna-
tha (Plecoptera). Spixiana 12(2): 145-182. https://www.biodiversi-
tylibrary. org/part/66924

Wang Y, Cao JJ, Li WH (2017) The complete mitochondrial genome of the
styloperlid stonefly species Styloperla spinicercia Wu (Insecta: Plecop-
tera) with family-level phylogenetic analyses of the Pteronarcyoidea.
Zootaxa 4243(1): 125-138. https://doi.org/10.11646/zootaxa.4243.1.5

Wang Y, Cao JJ, Li N, Ma GY, Li WH (2019) The first mitochondri-
al genome from Scopuridae (Insecta: Plecoptera) reveals structural
features and phylogenetic implications. International Journal of Bi-
ological Macromolecules 122: 893-902. https://doi.org/10.1016/.
ybiomac.2018.11.019

Wichard W, Arens W, Eisenbeis G (1999) Atlas zur Biologie der
Wasserinsekten. Springer, Berlin, Heidelberg, 338 pp. https://doi.
org/10.1007/978-3-642-39452-2

Wipfler B, Klug R, Ge SQ, Bai M, Goébbels J, Yang XK, Hornschemey-
er T (2015) The thorax of Mantophasmatodea, the morphology of
flightlessness, and the evolution of the neopteran insects. Cladistics
31(1): 50-70. https://doi.org/10.1111/cla. 12068

Wipfler B, Letsch H, Frandsen PB, Kapli P, Mayer C, Bartel D, Buck-
ley TR, Donath A, Edgerly-Rooks JS, Fujita M, Liu S, Machida R,

dez.pensoft.net

Mashimo Y, Misof B, Niehuis O, Peters RS, Petersen M, Podsi-
adlowski L, Schiitte K, Shimizu S, Uchifune T, Wilbrandt J, Yan
E, Zhou X, Simon S (2019) Evolutionary history of Polyneoptera
and its implications for our understanding of early winged insects.
Proceedings of the National Academy of Sciences of the United
States of America 116(8): 3024-3029. https://doi.org/10.1073/
pnas.1817794116

Yoshizawa K (2011) Monophyletic Polyneoptera recovered by wing
base structure. Systematic Entomology 36(3): 377-394. https://dot.
org/10.1111/).1365-3113.2011.00572.x

Zhao MY, Huo QB, Du YZ (2020) Molecular phylogeny inferred from
the mitochondrial genomes of Plecoptera with Oyamia nigribasis
(Plecoptera: Perlidae). Scientific Reports 10: 20955. https://dot.
org/10.1038/s41598-020-78082-y

Zwick P (1973) Insecta: Plecoptera. Phylogenetisches System und Kat-
alog. Das Tierreich 94, i-xxxi1, 465 pp. De Gruyter, Berlin, New
Yolk.

Zwick P (1980) Plecoptera (Sternfliegen). Handbuch der Zoologie. De
Gruyter, Berlin, New Yolk, 115 pp.

Zwick P (2000) Phylogenetic system and zoogeography of the Ple-
coptera. Annual Review of Entomology 45: 709-746. https://doi.
org/10.1146/annurev.ento.45.1.709